Olfactory receptors (ORs) are seven transmembrane domain G protein-coupled receptors (GPCRs) that govern the sense of smell in the olfactory epithelium, and comprise the largest gene family in the genome (˜1000 OR genes in mice [1] and ˜300 [2] in humans). Although this family was first identified over 20 years ago [3], the majority of ORs remain orphan receptors, with no known ligand. This is due, in large part, to the fact that OR deorphanization is typically attempted using in vitro ligand screening assays in heterologous cell systems which require surface expression of the OR as a prerequisite for the assay (i.e., HEK293T cells or Xenopus oocytes) [4-7]. Unfortunately, many ORs do not traffic to the cell surface in heterologous cell systems; rather, they are retained in the ER and degraded [8-10], making ligand assignment impossible. To combat this problem, studies have utilized the co-expression of various accessory proteins and/or the addition of N-terminal tags [11-14]. For example, the addition of the first 20 amino acids of rhodopsin onto the N-terminus of ORs (Rho tag) enhances OR surface expression for a number of ORs [15]. Similarly, receptor transporting protein (RTP), originally identified as a potential chaperone for ORs [16,17], also enhances expression of multiple ORs. A recent study showed that the best surface expression was achieved [18] by co-expressing the short form of RTP (RTP1S) [19], Ric8b (a putative GEF) [20] and Gαolf (the G protein that couples to ORs in the olfactory epithelium) [21] with Rho-tagged ORs. While these tools have been beneficial to the field [5,15,16,18,20,22-24] and are the most reliable enhancers of OR surface expression available to date, their effects are not universal. Despite these developments, many ORs are still unable to reach the cell surface when heterologously expressed, and thus remain as orphan receptors.
As membrane proteins, ORs enter the biosynthetic pathway upon translocation into the endoplasmic reticulum (ER). Typically, this is accomplished co-translationally where a signal peptide serves to mediate ER translocation through the heterotrimeric Sec61 complex that forms a channel in the ER membrane[25]. While most GPCRs use one of their transmembrane domains (TMD) as a signal anchor sequence, a small subset of GPCRs and other TMD proteins (and all secretory proteins) have cleavable signal peptides which are found at the extreme N-terminus of the immature protein [26,27]. As their name implies, these cleavable signal peptides are not incorporated into the mature protein; rather they are cleaved off in the ER membrane upon translocation. While cleavable signal peptides do not have a conserved sequence, they do share characteristic features including a hydrophobic region flanked by polar amino acids [25,26].
Recently, the single-spanning membrane protein, Leucine Rich Repeat Containing 32 (LRRC32) was found to possess a leucine-rich 17-amino acid cleavable signal peptide (MRPQILLLLALLTLGLA (SEQ ID NO:3)) which is required for proper ER translocation and surface expression in both T regulatory cells (where it is natively expressed) as well as in HEK293T cells [28]. Because the addition of other cleavable signal peptides has been shown to enhance surface expression for some GPCRs in cell culture [29,30], we hypothesized that the addition the LRRC32 signal peptide may promote surface expression of ORs. Importantly, as signal peptides are cleaved off in the ER, the addition of such a tag would not affect the mature protein, preventing any potential alteration or interference with ligand binding. To assay whether the addition of a cleavable signal peptide could aid in OR surface expression, we added the 17 amino acid signal peptide from LRRC32 (which we named “Lucy,” for its leucine repeats) to the N-terminus of 15 diverse ORs (murine ORs from both Class I and Class II, representing 11 different subfamilies, as well as 2 human ORs) and assayed for surface expression. We also combined our Lucy tag with both the Rho tag and the best practice in OR trafficking (co-expression with accessory proteins RTP1S, Ric8b and Gαolf [18]) in order to assess the universal effects of this tag. Here we report that the Lucy tag, in combination with the Rho tag and the accessory proteins, promotes surface expression of all ORs tested, raising the possibility for wide-spread deorphanization.